1. Field of the Invention
The present invention relates generally to the fields of enzymology and cell biology. More particularly, it concerns methods for assessing phosphodiesterase function.
2. Description of Related Art
Phosphodiesterase-5 (PDE5), which specifically degrades cGMP, is the target of sildenafil (Pfizer tradename Viagra®) in causing penile erection (Boolell et al., 1996; Corbin & Francis, 1999). PDE5 is present in high levels in smooth muscle tissues, but it is also present in platelets and other tissues (Francis et al., 1990; Francis et al., 2001). PDE5 is believed to contain a single catalytic domain and regulatory domain on each of two subunits. The catalytic domain is highly specific for cGMP, and it is homologous to those of the other family members of the superfamily of cyclic nucleotide phosphodiesterases (PDE) (McAllister-Lucas et al., 1993). The regulatory domain contains two GAF domains (Aravind & Ponting, 1997; McAllister-Lucas et al., 1993; Thomas et al., 1990a) (GAF=cGMP-Anabaena adenylate cyclase-Escherichia coli FhIA), at least one of which functions as a highly selective allosteric site for cGMP binding (Liu et al., 2001a; McAllister-Lucas et al., 1993). Four other PDE families (PDE2, PDE6, PDE10, and PDE11) also possess GAF domains (Charbonneau, 1990; Charbonneau et al., 1990; Fawcett et al., 2000; Francis et al., 2001; Fujishige et al., 1999; Soderling et al., 1999). In addition to PDE5, two other PDE families (PDE2, PDE6) have been shown to bind cGMP (Stroop & Beavo, 1991; Yamazaki et al., 1980).
Binding of cGMP to the GAF domains of PDE2 stimulates catalytic activity of this enzyme (Beavo et al., 1971). The regulatory domain in PDE5 is phosphorylated by cGMP-dependent protein kinase (PKG) at Ser-92 (bovine) both in vitro and in intact cells (Mullershausen et al., 2001; Murthy, 2001; Rybalkin et al., 2002; Thomas et al., 1990b; Wyatt et al., 1998). Regulation of phosphorylation of this site is substrate-directed since occupation of a GAF domain by cGMP is required for phosphorylation by PKG. Phosphorylation causes stimulation of both catalytic activity and cGMP binding to the GAF domain (Corbin et al., 2000; Francis et al., 2002).
It has been proposed that these effects are responsible for negative feedback regulation of active cGMP levels in cells. For both the cGMP and cAMP signaling pathways, negative feedback regulation has emerged as a major function of PDEs. PDE3 and PDE4 have been reported to be activated by phosphorylation when cAMP is elevated in cells (Conti, 2000; Degerman et al., 1997). Likewise, elevation of cGMP has been shown to cause increased PDE5 activity that is associated with cGMP-sensitive phosphorylation (Mullershausen et al., 2001; Murthy, 2001; Wyatt et al., 1998). In each case, PDE phosphorylation correlates with increased degradation of cyclic nucleotide. Elevation of cGMP is required for phosphorylation of PDE5 by PKG or cAMP-dependent protein kinase (PKA), and this process stimulates degradation of cGMP as well as sequestration of cGMP in the GAF domain (Corbin et al., 2000), both of which represent negative feedback on the cGMP pathway.
Negative feedback regulation of cGMP would be enhanced if cGMP binding to the GAF domain also directly stimulates the catalytic domain. This was predicted earlier from the principle of reciprocity (Thomas et al., 1990a; Weber, 1975; Francis et al., 1990). Binding of 3-isobutyl-1-methylxanthine (IBMX) or a similar ligand to the catalytic domain has been shown to stimulate binding of cGMP to a GAF domain, but direct evidence that binding of cGMP to a GAF domain stimulates the catalytic domain has been elusive. This has been due in large part to difficulties in performing such studies with PDE5, in which both the catalytic domain and GAF domain possess high specificity for cGMP. Okada & Asakawa recently reported that cGMP stimulates PDE5 catalytic activity when measured using a fluorescent cGMP analog that is specific for the catalytic site of the enzyme (Okada & Asakawa, 2002). They suggested that this stimulation occurs through cGMP binding to the GAF domains.
Studies of the catalytic domain of PDE5 would be greatly enhanced by a specific, high-affinity PDE5 catalytic-site radioligand that binds with sufficient affinity to allow isolation of the ligand-enzyme complex. In addition, binding assays based on this interaction could be used to screen for potentially efficacious PDE inhibitors. Properties of the catalytic site that are not directly dependent on catalytic activity could also be examined using such a ligand.